EP1587945A2 - Hydroxyphenyl cross-linked macromolecular network and applications thereof - Google Patents
Hydroxyphenyl cross-linked macromolecular network and applications thereofInfo
- Publication number
- EP1587945A2 EP1587945A2 EP04701177A EP04701177A EP1587945A2 EP 1587945 A2 EP1587945 A2 EP 1587945A2 EP 04701177 A EP04701177 A EP 04701177A EP 04701177 A EP04701177 A EP 04701177A EP 1587945 A2 EP1587945 A2 EP 1587945A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- hydroxyphenyl
- macromolecular network
- hydrogel
- molecules
- substituted
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/78—Preparation processes
- C08G63/81—Preparation processes using solvents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L101/00—Compositions of unspecified macromolecular compounds
- C08L101/12—Compositions of unspecified macromolecular compounds characterised by physical features, e.g. anisotropy, viscosity or electrical conductivity
- C08L101/14—Compositions of unspecified macromolecular compounds characterised by physical features, e.g. anisotropy, viscosity or electrical conductivity the macromolecular compounds being water soluble or water swellable, e.g. aqueous gels
Definitions
- Articular cartilage performs an essential function in healthy joints. It is responsible for absorbing and dissipating impact and frictional loads in order to divert these loads away from bones, to protect the bones from damage. Cartilage performs this function by transferring the loading force to a fluid phase within a three-dimensional network of aggrecan molecules, themselves constrained (described in the next paragraph) within the joint space.
- Aggrecan molecules have up to 100 chondroitin sulfate chains attached to a core protein, with each chondroitin sulfate chain possessing multiple negatively charged sulfate groups along their length.
- the effect of all these sulfate groups is to cause each of the chondroitin sulfate chains in a single aggrecan molecule to repel one another, (resulting in the aggrecan molecule having the maximum possible volume at rest), and also to cause adjacent aggrecan molecules in a cartilage aggregate to repel one another.
- aggrecan molecules are attached to long hyaluronan chains, which are in turn constrained in large cartilage aggregates within the joint space by an extracellular collagen fibril matrix.
- adjacent chondroitin sulfate chains in each aggrecan molecule repel one another, they are nonetheless constrained within the collagen matrix. See Fig. 1 depicting normal, healthy cartilage.
- the hyaluronan-aggrecan network expands as much as possible within the constraints of the collagen matrix to achieve the lowest possible energy state at rest; i.e.
- the macromolecular network diverts impact and frictional loads away from bones by transferring them to the continuous fluid (water) phase as follows.
- the force is absorbed first by the macromolecular network, where it acts on and tends to deform or compress the network.
- the force sets up pressure gradients in the fluid phase in order to induce fluid flow to accommodate network deformation or compression resulting from the load.
- the fluid cannot negotiate the tight macromolecular network, packed with the repulsive chondroitin sulfate chains, sufficiently to accommodate a bulk flow of water without shifting or displacing the network molecules.
- Osteoarthritis and rheumatoid arthritis affect an estimated 20.7 and 2.1 million Americans, respectively. Osteoarthritis alone is responsible for roughly 7 million physician visits a year. For severe disabling arthritis, current treatment involves total joint replacement with on average 168,000 total hip replacements and 267,000 total knee replacements performed per year in the U.S. alone. Defects in articular cartilage present a complicated treatment problem because of the limited capacity of chondrocytes to repair cartilage. Treatment strategies to date have focused on the use of autologous chondrocytes expanded in culture or the recruitment of mesenchymal stem cells in vivo by chemotactic or mitogenic agents.
- Hyaluronan has been proposed as a candidate for the development of biomaterials for local delivery of chondrocytes or bioactive agents because of its unique properties, including excellent biocompatibility, degradability, and rheological and physiochemical properties.
- chondrocytes suspended in a tissue engineered hyaluronan matrix would be able to synthesize a new cartilage matrix with mechanical properties comparable to normal, healthy articular cartilage. This is because conventional biomaterials made from hyaluronan are formed through chemistries that are incompatible with maintaining cell viability. Chondrocytes must be introduced to the matrices after matrix formation with variable and normally poor results.
- an artificial or synthetic matrix that can effectively divert a loading force from bones in an effective manner.
- a matrix can be provided in situ or in vivo to repair or replace articular cartilage during an orthopedic surgical procedure.
- the artificial or synthetic matrix can be provided to an in situ or in vivo target site as a liquid or a plurality of liquids, and can set up in place to provide a substantially seamless integration with existing cartilaginous and or bony tissue in a patient.
- a macromolecular network including the following structure
- R t and R 2 each is or includes a structure selected from the group consisting of polycarboxylates, polyamines, polyhydroxyphenyl molecules, and copolymers thereof, and wherein Rt and R 2 can be the same or different structures.
- a macromolecular network having a plurality of tyramine-substituted hyaluronan molecules, wherein at least two adjacent hyaluronan molecules are linked by a dityramine linkage.
- a hydrogel is also provided which includes a macromolecular network of tyramine- substituted hyaluronan molecules that are cross-linked by dityramine linkages between hyaluronan molecules.
- a method of making a macromolecular network including the steps of providing a first macromolecular species selected from the group consisting of hydroxyphenyl-substituted polycarboxylates, hydroxyphenyl-substituted polyamines, other polyhydroxyphenyl molecules, and copolymers thereof, and forming at least one dihydroxyphenyl linkage between two hydroxyphenyl groups attached respectively to adjacent ones of the first macromolecular species.
- a method of making a hydrogel having the following steps: a) providing a first solution having either a peroxidase enzyme or a peroxide but not both, and also a macromolecular species selected from the group consisting of hydroxyphenyl- substituted polycarboxylates, hydroxyphenyl-substituted polyamines, other polyhydroxyphenyl molecules, and copolymers thereof; b) providing a second solution having the one of the peroxidase enzyme or peroxide not provided in the first solution; and c) combining the first and second solutions to initiate dihydroxyphenyl cross-linking to form the hydrogel.
- Fig. 1 is a schematic diagram of normal, healthy human cartilage.
- Fig. 2 is a schematic diagram of a dihydroxyphenyl cross-linked macromolecular network according to the invention.
- Fig. 3 is a structural formula of a hyaluronan molecule.
- Fig. 4 is a graph showing comparative results for mechanical testing in a confined compression test (equilibrium stress versus applied strain) of T-HA hydrogels according to the invention versus published results for articular cartilage plugs (Example 3).
- Fig. 5 is a graph showing comparative data of glucose utilization for chondrocytes embedded in T-HA hydrogels (1.7% and 4.7% T-HA) compared to cultured on tissue culture plastic (control).
- polycarboxylate means a molecule, structure or species having a chain length of at least two functional groups or units, wherein at least two such groups or units of the chain are or comprise carboxylic acid groups that are sterically accessible to a nucleophilic substitution reaction as described herein.
- polyamine means a molecule, structure or species having a chain length of at least two functional groups or units, wherein at least two such groups or units of the chain are or comprise primary amine groups that are available for a nucleophilic substitution reaction.
- a polyhydroxyphenyl molecule means a molecule having a chain length of at least two functional groups or units, wherein at least two such groups or units of the chain are or comprise hydroxyphenyl groups that can be linked to another hydroxyphenyl group via a C-C bond.
- a hydrogel is a material that is prepared comprising a macromolecular network that is used or useful in tissue replacement or engineering applications, e.g. as artificial cartilage, as a material to coat surgical instruments to prevent tissue irritation, or to provide a semi-permeable membrane such as for use in an artificial kidney, etc.
- the invention includes a novel structure of a macromolecular network that has been formed by linking hydroxyphenyl groups attached to adjacent long chain macromolecules, resulting in effectively cross-linking the macromolecules to provide a large network.
- the basic cross-linking structure of the network is shown below
- Ri and R 2 are each long chain macromolecules. Ri and R can be the same molecule or different molecules, but it will be understood that to provide a suitable network, Ri and R 2 will be different molecules for at least a portion of the dihydroxyphenyl linkages in a network according to the invention. It is not necessary, though it is preferred, that R t and R 2 are the same species of molecule.
- a network of dihydroxyphenyl cross-linked macromolecules is provided as shown schematically in Fig. 2.
- the macromolecules are represented schematically by cylindrical strands, each having at least two hydroxyphenyl groups attached along its length. It is noted that not every hydroxyphenyl group must be linked to another hydroxyphenyl group.
- the disclosed invention involves covalent coupling of hydroxyphenyl containing compounds, including but not limited to tyramine, through their primary amine (or carboxyl) groups to carboxyl (or primary amine) groups on various polymeric scaffold materials, including but not limited to hyaluronan or chondroitin sulfate (e.g. in the form of aggrecan), via a carbodiimide-mediated reaction.
- hydroxyphenyl residues are selectively cross-linked by horseradish peroxidase (HRP) in the presence of very dilute hydrogen peroxide to form hydrogels.
- HRP horseradish peroxidase
- the first step in providing the macromolecular network is to prepare or provide the long-chain macromolecules having periodic hydroxyphenyl groups attached.
- the macromolecules are polyhydroxyphenyl molecules which already have multiple or periodic hydroxyphenyl groups, such as polyphenols.
- Suitable polyphenols include polyamino acids (e.g. polytyrosine), epigallocatechin (EGC), and epigallocatechin gallate (EGCG) isolated from green tea, less preferably other polyphenols.
- the hydroxyphenyl groups can be added to the macromolecules periodically or randomly along their length via a chemical reaction.
- a preferred method of adding hydroxyphenyl groups to the macromolecules is to utilize a carbodiimide-mediated substitution reaction pathway to provide an amide bond between a primary amine having a hydroxyphenyl group and a carboxylic acid group attached to the macromolecules.
- the long-chain macromolecule preferably is a polycarboxylate molecule, having periodic carboxylic acid groups along its length.
- the hydroxyphenyl groups are provided as part of smaller molecules having primary amine groups that can be attached to the carboxyl carbon atoms of a carboxylic acid group on the long-chain macromolecules via the carbodiimide pathway. The reaction proceeds as follows:
- Structure A is a carbodiimide
- Structure B is a polycarboxylate (though only one CO 2 H group is shown);
- Structure C is the product of Reaction A and is an activated O-acylisourea
- Structure D is a primary amine having a hydroxyphenyl group
- Structure E is a hydroxyphenyl-substituted polycarboxylate
- Structure F is an acylurea byproduct; wherein individual Rs can be individually selected, the same or different from one another, to be a straight chain or branched alkane or acyl group, or any other structure that does not interfere with the carbodiimide reaction pathway to provide the amide bond between the NH 2 and CO 2 H groups as shown in Structure E above.
- Reaction A represents a carbodiimide activation of the carboxyl group to provide an activated O-acylisourea intermediate.
- the electropositive carbon atom of this intermediate is receptive to nucleophilic attack by the lone pair of electrons on a nitrogen atom of an adjacent primary amine molecule having an attached hydroxyphenyl group.
- the products of this nucleophilic substitution reaction (Reaction B) are a hydroxyphenyl-substituted polycarboxylate and an acylurea byproduct which can be dialyzed out to provide a substantially pure hydroxyphenyl-substitute polycarboxylate product.
- the carbodiimide can react with nucleophiles other than the carboxylate oxygen atom of the polycarboxylate molecule required to form the desired O-acylisourea (reaction A).
- nucleophiles may include the amine and/or hydroxyphenyl groups of Structure D illustrated above.
- reaction C The product of an amine reaction with the carbodiimide (Reaction C) will not have a free amine group effectively reducing the amount of tyramine available for reaction with the O- acylisourea. This reaction also reduces the amount of carbodiimide available for formation of the desired O-acylisourea.
- the products of the hydroxyphenyl reaction (Reaction D) are not UN absorbent, which will make their detection by UV-spectroscopy in the final hydroxyphenyl-substituted polycarboxylate product (explained below) more difficult. However, because these products still contain free amine groups, they can form amide bonds with the polycarboxylate molecule via Reaction B.
- the O-acylisourea (Structure C) can be hydrolyzed as shown in Reaction F releasing the original unmodified polycarboxylate (Structure B) and the acylurea of the carbodiimide (Structure F). This is an unproductive reaction similar to reaction E, which reduces the effective concentration of the carbodiimide.
- the O-acylisourea can also undergo an intramolecular rearrangement (Reaction G) to form two unreactive N-acylureas. These structures form unproductive adducts on the carboxylate molecule which cannot contribute to the peroxidase catalyzed cross-linking reaction shown (step 2 discussed below) for preparing the network according to the invention.
- the O-acylisourea can also react (Reaction H) with a second carboxyl group on either the same or a different polycarboxylate molecule to form an acid anhydride. This molecule can then react with Structure D to form the desired amide and regenerate the second carboxyl group.
- reaction H reacts with a second carboxyl group on either the same or a different polycarboxylate molecule to form an acid anhydride.
- This molecule can then react with Structure D to form the desired amide and regenerate the second carboxyl group.
- the macromolecule can be a polyamine having multiple or periodic amine groups along its length, wherein the hydroxyphenyl groups then are provided as part of smaller carboxylic acid molecules.
- Suitable polyamines include: polyhexosamines such as chitosan (polyglucosamine); polyamino acids such as polylysine; polydeoxyribonucleotides such as poly (dA) (polydeoxyadenylic acid), poly(dC) (polydeoxycytidylic acid), and poly(dG) (porydeoxyguanylic acid); and polyribonucleotides such as poly(A) (polyadenylic acid), poly(C) (polycytidylic acid), and poly(G) (polyguanylic acid).
- polyhexosamines such as chitosan (polyglucosamine); polyamino acids such as polylysine
- polydeoxyribonucleotides such as poly (dA) (polydeoxyadenylic acid), poly(dC) (polydeoxycytidylic acid), and poly(dG) (porydeoxy
- the carbodiimide- mediated reaction pathway proceeds exactly as explained above to form the amide bond between the amine group and carboxylic acid group except that, as will be understood by a person having ordinary skill in the art, the resulting product will be hydroxyphenyl- substituted polyamine instead of a polycarboxylate.
- Other peptides and/or proteins also can be used as the macromolecules in the present invention, either which have hydroxyphenyl groups disposed along their length, or to which hydroxyphenyl groups can be provided via a substitution reaction as described herein.
- polyarginrne can be used as the macromolecule.
- suitable hydroxyphenyl- containing compounds for use in the present invention include those having a free primary amine that can be used to modify scaffold materials having multiple or periodic CO H groups, including tyrosine (2-amino-3-(4-hydroxyphenyl) proprionic acid) and tyramine (tyrosamine or 2-(4-hydroxyphenyl) ethylamine).
- suitable hydroxyphenyl-containing compounds include those having a free CO 2 H group that can be used to modify scaffold materials having multiple or periodic primary NH 2 groups, including tyrosine, 3-(4-hydroxyphenyl) propionic acid and 4-hydroxyphenylacetic acid.
- the second step in preparing the cross-linked macromolecular network according to the invention is to link the resulting macromolecules, now having one or more hydroxyphenyl groups attached, via a dihydroxyphenyl linking structure.
- hydroxyphenyl groups attached to different macromolecules are linked via the reaction mechanism shown below using a peroxide reagent in the presence of a peroxidase:
- Peroxidase in the presence of a dilute peroxide is able to extract the phenolic hydroxyl hydrogen atom from hydroxyphenyl containing compounds (such as tyramine) leaving the phenolic hydroxyl oxygen with a single unshared electron, an extremely reactive free radical.
- the free radical isomerizes to one of the two equivalent ortho-position carbons and then two such structures dimerize to form a covalent bond effectively cross-linking the structures, which after enolizing generates a dihydroxyphenyl dimer (a dihydroxyphenyl linkage such as dityramine linkage as described below).
- reaction conditions peroxide and peroxidase.
- Hydrogen peroxide is indicated in the above mechanism, but other suitable peroxides can be used.
- the peroxidase preferably is horseradish peroxidase (HRP).
- HRP horseradish peroxidase
- any other suitable enzyme or other agent can be used that is capable of generating free-radicals for cross-linking scaffold materials containing hydroxyphenyl groups, preferably under ordinary metabolic conditions as described below.
- the dihydroxyphenyl cross-linked macromolecular network is superior to conventional cartilage or other tissue replacement or substitution methods and products because the cross-linking reaction is enzyme driven (peroxidase).
- This means the cross- linking reaction is carried out under ordinary in vivo or metabolic conditions of temperature such as 35-39°C (e.g. about 37°C), pH range of 6-7 (e.g. about 6.5), reagents etc.
- a peroxide, such as hydrogen peroxide, is the only required reagent for the cross-linking reaction).
- the cross-linking reaction can be performed in vivo, to provide a cross-linked hydrogel at a surgical situs, such as an orthopedic surgical situs, to promote maximum seamless integration between the hydrogel and native tissue such as bony and cartilaginous tissue.
- Integration of the new hydrogel scaffold with native cartilage matrix may occur immediately as the hydroxyphenyl-substituted macromolecular scaffold quickly penetrates into the existing cartilage matrix prior to cross-linking, and cross-links not only with other hydroxyphenyl-substituted macromolecular scaffold material but potentially with tyrosine residues of resident proteins in the existing cartilage matrix. This would eliminate a typical problem found with pre-formed matrix plugs, which is their poor integration into the native cartilage tissue.
- solutions containing all but one of these components can be prepared for convenient application to a surgical site.
- a solution comprising a tyramine - (or other hydroxyphenyl containing species) substituted polycarboxylate (such as tyramine-substituted hyaluronan, etc.) and the peroxidase can be prepared, with a second solution prepared containing the peroxide.
- the peroxide and the peroxidase can be swapped between the first and second solutions, the important thing being that the peroxide and peroxidase are kept separate (i.e.
- the cross-linking reaction occurs in vivo.
- the first solution is applied, (e.g. to an in vivo surgical situs), and the second solution is applied or sprayed over the first, in vivo, to cause in situ cross-linking of the tyramine residues.
- the cross linking reaction occurs in vivo.
- additional living cells such as chondrocytes, progenitor cells, stem cells, etc.
- additional living cells such as chondrocytes, progenitor cells, stem cells, etc.
- chondrocytes, progenitor cells, stem cells, etc. can be provided directly to a medium containing the non-cross-hnked hydroxyphenyl-substituted polycarboxylates or polyamines (or polyphenols), i.e. to the first or second solution from the preceding paragraph, wherein the cell-rich medium is applied with the macromolecules to the site in vivo, and the molecules are subsequently cross-linked via addition of peroxidase and peroxide.
- the result is a cross-linked macromolecular network containing the desired cells dispersed within it.
- the macromolecule used to produce the network according to the invention is hyaluronan or hyaluronic acid (HA), and the hydroxyphenyl group is supplied in the form of tyramine.
- HA is composed of repeating pairs of glucuronic acid (glcA) andN- acetylglucosamine (glcNAc) residues linked by a ⁇ 1,3 glycosidic bond as shown in Fig. 3. For each hyaluronan chain, this simple disaccharide is repeated up to 10,000 times with each repeat disaccharide linked by a ⁇ 1 ,4 glycosidic bond.
- Each glcA residue has a carboxylic acid group (CO 2 H) attached to the number 5 carbon atom of the glucose ring.
- Tyramine is a phenolic molecule having an ethyl amine group attached para to the OH group on the benzene ring.
- the mechanism for tyramine substitution onto the singly bound oxygen atom of a CO 2 H group on HA proceeds via the carbodiimide-mediated reaction mechanism described above as illustrated immediately below.
- the preferred carbodiimide species is l-ethyl-3-(3-dimethylammopropyl)carbodiimide (EDC) as shown.
- Structure A is EDC
- Structure B is hyaluronan (though only one CO 2 H group is shown); Structure C is the product of Reaction A and is l-ethyl-3-(3-dimethylaminopro ⁇ yl) isourea;
- Structure D is tyramine
- Structure E is tyramine-substituted hyaluronan
- Structure F is l-ethyl-3-(3-dimethylaminopropyl) urea (EDU).
- Reaction A a negatively charged oxygen atom of the carboxyl group of the hyaluronan molecule attacks, via a nucleophilic reaction mechanism, the electron-deficient diimide carbon atom on the carbodiimide molecule (EDC) to form the activated O- acylisourea (Reaction A).
- EDC electron-deficient diimide carbon atom on the carbodiimide molecule
- Reaction B the activated O- acylisourea
- Reaction B Reaction A is preferably catalyzed by a suitable catalyst that will result in the formation of an active ester during Reaction A, thus permitting the reaction to be carried out at substantially neutral pH (e.g.
- Suitable catalysts include N-hydroxysuccmimide (NHS), less preferably 1-hydroxybenzotriazole (HOBt) orN-hydroxysulfosuccinimide (NHSS), less preferably another suitable catalyst or combinations thereof effective to enhance the carbodiimide reaction by formation of an active ester in order to minimize the unproductive hydrolysis of carbodiimides at higher pHs.
- NHS N-hydroxysuccmimide
- HOBt 1-hydroxybenzotriazole
- NHSS N-hydroxysulfosuccinimide
- NHSS N-hydroxysuccmimide
- CMC l-cyclohexyl-3-[2-(4-methylmorpholmo)ethyl]carbodiimide
- DCC dicyclohexylcarbodiimide
- Reaction A O-acylisourea-substituted hyaluronan; essentially the EDC molecule has been temporarily substituted onto the carboxylic acid group of a glcA residue from the HA molecule, making the carbon atom of the carboxylic acid group slightly positively charged.
- the electron pair from the terminal amine group of a tyramine molecule is then substituted onto the carbon atom via a nucleophilic substitution reaction as explained in the preceding paragraph (Reaction B).
- the result of Reaction B is the tyramine-substituted HA molecule (T-HA) and acylurea, a byproduct. It will be understood that Reactions A and B will result in a plurality of tyramine substitutions on the periodic glcA residues of HA molecules; a single substitution has been shown here for brevity and clarity.
- T-HA After formation of T-HA, a plurality of T-HA molecules are reacted via peroxide and peroxidase enzyme to cross-link T-HA molecules as previously described and illustrated above. That is, the hydroxyphenyl groups on the tyramine residues now attached to HA molecules react with peroxide (preferably H 2 O 2 ) in the presence of a peroxidase to remove the phenolic hydrogen atom resulting in a tyramine free radical, with the unpaired electron associated with the phenolic oxygen atom.
- peroxide preferably H 2 O 2
- This free radical species isomerizes or resonates, resulting in a resonance structure (or free radical isomer) with the unpaired electron now associated with an ortho carbon atom on the phenolic ring.
- the cross-linked T-HA network can be provided with aggrecan molecules in a conventional manner, e.g. via link proteins, to provide a cross-linked T-HA network having aggrecan molecules attached to the HA chains.
- a network similar to that found in a normal cartilage aggregate can be provided according to the invention, with the dityramine bonds holding the network together thereby constraining the contained aggrecan network, instead of collagen fibrils as in normal cartilage.
- glycosaminoglycans other glycosaccharides and polycarboxylic acids
- suitable glycosaminoglycans include chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin.
- polycarboxylates include: proteoglycans such as versican, aggrecan, and cartilage aggregates composed of aggrecan, hyaluronan and link protein; polyuronic acids such as polypectate (polygalacturonic acid), polyglucuronic acid, pectin (polygalacturonic acid methyl ester), colominic acid ( oly[2,8-(N-acety euraminic acid)]), and alginate (poly[mannuronate-co-guluronate]); and amino acids (having at least 2 amino acid units) that meet the definition of polycarboxylate given above, such as polyaspartic acid, and polyglutamic acid. All of these can be substituted with one or a plurality of hydroxyphenyl groups using the carbodiimide-mediated reaction pathway disclosed herein by a person of ordinary skill in the art without undue experimentation.
- native polyphenol compounds which already contain two or more hydroxyphenyl groups that can be cross-linked using the described enzyme catalysis chemistry can be used in place of the polycarboxylates and polyamines described above which must have the hydroxyphenyl groups added by a chemical reaction.
- a network of tyramine cross-linked chondroitin sulfate molecules (either alone or provided as part of aggrecans) is provided to simulate or replace normal cartilage.
- Chondroitin sulfate is identical to hyaluronan except: 1) the repeat disaccharide structure contains N-acetylgalactosamine (galNAc) rather than glcNAc, a difference in only the position of the hydroxyl group attached to the 4- carbon (circled in Fig. 3); 2) the presence of O-sulfation on the hydroxyl groups at the 4- and/or 6-position of the galNAc residue and/or the 2-position of the glcA residue (Fig.
- chondroitin sulfate chains which are smaller than hyaluronan with between 20 to 100 repeating disaccharide units.
- An aggrecan molecule is made up of multiple - roughly 100 chondroitin sulfate chains linked to a core protein through a linkage saccharide located at each chain's reducing end).
- the negatively charged SO 2" groups of adjacent (cross-linked) chondroitin sulfate molecules provide the principal repulsive force contributing to the compression resistance of the network aggregate while the tyramine crosslinks constrain the chondroitin sulfate network from breaking or dissipating.
- cartilage degenerative conditions are entirely circumvented; e.g. conditions where the core protein to which chondroitin sulfate molecules are ordinarily bonded in normal cartilage becomes cleaved between the HA binding domain (Gl) and the second globular domain (G2) thus allowing the chondroitin sulfate rich region to diffuse out from the cartilage aggregate.
- the chondroitin sulfate molecules are directly cross-linked to one another, unassociated with an aggrecan or other proteoglycan molecule, they cannot be cleaved or carried away as in normal cartilage.
- a tyramine cross-linked T-HA network (having an HA backbone chain with attached aggrecan molecules, which in turn include chondroitin sulfate chains) may be preferred because of the high availability of HA. This may be beneficial in the case of cartilage replacement or repair using the present invention, because the body's normal metabolic pathway for generating cartilage may be able to build directly onto an implanted tyramine cross-linked T-HA network as will be described.
- the dityramine cross-linked T-HA network described above has particular utility for producing artificial or synthetic cartilage.
- Cartilage implants are frequently used in reconstructive procedures of the head and neck to repair cartilaginous or bony defects secondary to trauma or congenital abnormalities.
- Applications specific to the ear include otoplasty and auricular reconstruction, which are often undertaken to repair cartilaginous defects due to trauma, neoplasm (i.e., squamous cell carcinoma, basal cell carcinoma, and melanoma), and congenital defects such as microtia.
- Applications specific to the nose include cosmetic and reconstructive procedures of the nose and nasal septum. Dermasal hump augmentation, tip, shield and spreader grafts are frequently used in cosmetic rhinoplasty.
- MEMS devices have been designed for use in artificial kidney devices, which contain precisely defined micropores that can effectively mimic only the size exclusion characteristics of the kidney.
- the charge exclusion related filtration is the result of heparan sulfate proteoglycans present in a basement membrane, which separates two distinct cell types important for other kidney related functions.
- hydrogels can be prepared composed of either heparan sulfate or heparin that are cross-linked via dihydroxyphenyl (dityramine) links as described herein and provided within the pores of the MEMS device.
- This heparin heparan sulfate hydrogel can then be sandwiched between two hyaluronan derived hydrogels (e.g. T- HA described above) as described herein, and containing one of each of the cell types normally found in a normally functioning kidney.
- the central heparinheparan sulfate hydrogel provides the charge exclusion properties for the device.
- the outer two hyaluronan hydrogel layers provide protection from the immune system and fouling by normal cellular and molecular debris. Inclusion of the two cell types on opposite sides of the filtration barrier provides a cellular component in its normal physiologic orientation.
- the hydrogels according to the invention can be applied in developing an artificial pancreas.
- a problem in development of an artificial pancreas is the short half life of MEMS engineered glucose sensors due to fouling of the detector electrode in vivo.
- Coating of the surface of these detectors with a hyaluronan hydrogel (e.g. T-HA) as described herein would permit diffusion of the small molecular weight glucose molecules that they are designed to detect while providing protection from the immune system and fouling by normal cellular and molecular debris.
- macromolecules useful as scaffold materials for formation of hydrogels include but are not limited to polycarboxylates (containing free carboxylate groups), polyamines (containing free primary amine groups), polyphenols (containing free hydroxyphenyl groups) and their copolymers, examples of which have been described above.
- polycarboxylates containing free carboxylate groups
- polyamines containing free primary amine groups
- polyphenols containing free hydroxyphenyl groups
- their copolymers examples of which have been described above.
- the first step in preparing the network according to the invention described above can be omitted because polyphenols already contain multiple or periodic hydroxyphenyl groups. Otherwise, both polycarboxylates and polyamines must have hydroxyphenyl groups added or substituted along their length, preferably via the above-described carbodiimide reaction pathway.
- the second step in preparing the network is to carry out an enzyme driven dimerization reaction between two hydroxyphenyl groups attached to adjacent macromolecules (whether polycarboxylates, polyamines or polyphenols) in order to provide a cross-linked structure.
- This step is carried out using a peroxide reagent (preferably hydrogen peroxide) in the presence of a suitable enzyme (preferably HRP) under metabolic conditions of temperature andpH.
- the carboxyl groups on high molecular weight hyaluronan (HA) are substituted with tyramine which introduces reactive hydroxyphenyl groups into the HA molecule.
- This tyramine substitution reaction preferably is mediated by the carbodiimide, l-ethyl-3-(3- dimethylammopropyl)carbodiimide (EDC) with the degree of tyramine substitution on HA controlled by the molar ratios and absolute concentrations of tyramine, EDC and HA used in the reaction mix.
- T-HA high molecular weight tyramine- substituted HA
- the percent tyramine substitution within each T-HA preparation is easily calculated by measuring: 1) the concentration of tyramine present in the preparation, which is quantitated specfrophotometrically based on the unique UV-absorbance properties of tyramine at 275 nm (see Example 2 below); and 2) the concentration of total carboxyl groups in the HA preparation, which is quantitated spectrophotometrically by a standard hexuronic acid assay.
- T-HA preparations which contain a percent tyramine substitution of only 4 - 6% have been routinely synthesized experimentally.
- the vast majority preferably at least 60, 70, 80, 90, or 95, percent
- the vast majority preferably at least 60, 70, 80, 90, or 95, percent
- solutions of T-HA are cross-linked to form hydrogels through an enzyme (peroxidase) driven reaction, which catalyzes the formation of a covalent bond between two tyramine adducts on adjacent HA molecules, producing a single dityramine cross-link.
- enzyme peroxidase
- the formation of hundreds of these dityramine cross-links per HA molecule result in formation of a stable 3-dimentional scaffold or hydrogel.
- Addition of very dilute peroxide preferably H 2 O
- the products of the reaction of the peroxidase enzyme on peroxide are free radicals that are preferentially taken up by the hydroxyphenyl rings of tyramine resulting in the formation of the dityramine crosslinks.
- the dityramine linked structures are fluorescent blue (see Example 2), a property which is used to both image the hydrogels and to quantify the degree of cross-linking within the hydrogels. Since the cross-linking reaction is enzyme driven, the hydrogels can be formed under physiologic conditions, and therefore can be formed in the presence of included cells or bioactive agents, or directly adjacent to living tissue while mamtaining cell and tissue viability.
- hydrogels are optically clear with a wide range of physical properties depending on the initial T-HA concentration.
- hydrogels formed from T-HA solutions of 6.25, 12.5, 25, 50 and 100 mg/ml T-HA have been shown experimentally to have physical properties (rigidity, rheology and texture) of a jelly, a gelatin, a dough, a resilient rubber-like composition (similar to a rubber ball), and a cartilage-like material respectively - see Example 3.
- T-HA solutions 6.25, 12.5, 25, 50 and 100 mg/ml T-HA
- These materials have potential applications in a wide range of clinical settings including tissue engineering of both orthopedic (i.e.
- non-orthopaedic kidney, liver, pancreas, etc. tissues, gene and drug delivery, coating of non-biological devices for in vivo implantation (i.e. glucose sensors, artificial hearts, etc.), wound repair, biosensor design, and vocal chord reconstruction.
- Advantageous properties of the hydrogels described herein include the ability to: 1) provide easy characterization and quality control; 2) integrate with existing tissue matrices; 3) directly incorporate into newly formed matrices; 4) directly include cells and bioactive factors; 5) maintain biocompatibility; 6) control bioresorption; 7) cast easily into complicated anatomical shapes (see Example 6 below); and 8) exhibit the mechanical properties of native tissues such as articular cartilage.
- tyramine-substituted hyaluronan hydrogels having dityramine cross-links according to the invention have been prepared as follows.
- HA is dissolved at 1 mg/ml based on hexuronic acid in 250 mM 2-(K-mo holino)ethanesulfonic acid (MES), 150 mMNaCl, 75 mMNaOH, pH 6.5 containing a 10-fold molar excess of tyramine relative to the molar concentration of HA carboxyl groups.
- Tyramine substitution onto the carboxyl groups is then initiated by the addition of a 10-fold molar excess of EDC relative to the molar concentration of the HA carboxyl groups.
- a 1/lOth molar ratio of N- hydroxysuccinimide (NHS) relative to the molar amount of EDC is added to the reactions to assist the EDC catalyzed amidation reaction by formation of active esters. Reactions are carried out at room temperature for 24 hours, after which the macromolecular fraction is recovered from unreacted small molecular weight reactants such as tyramine, EDC, NHS, and MES by exhaustive dialysis versus 150 mMNaCl and then ultrapure water followed by lyophilization.
- the tyramine-substituted HA (T-HA) product is dissolved to working concentrations of between 5 and 100 mg/ml in PBS (which is a buffer compatible with cell suspension, in vivo tissue contact, and the cross-linking reaction) to provide various concentration preparations depending on the desired rigidity of the final hydrogel.
- PBS which is a buffer compatible with cell suspension, in vivo tissue contact, and the cross-linking reaction
- the solvent can be any other suitable solvent besides PBS that will not substantially negatively impact the enzyme activity and that will not interfere with cross- linking reaction via selective uptake of free radicals generated by the enzyme.
- Suitable alternative solvents include water, conventional biological tissue culture media, and cell freezing solution (generally composed of about 90% blood serum and about 10% dimethyl sulfoxide).
- T- HA Prior to suspension of cells (see Example 5) or contact with tissues in vivo, the T- HA should be filtered through a 0.2 ⁇ m filter.
- tyramine-tyramine linking is carried out by adding 10 U/ml of type II horseradish peroxidase (HRP) to each T-HA preparation.
- HRP horseradish peroxidase
- Cross-linking is initiated by the addition of a small volume (1-5 ⁇ l) of a dilute hydrogen peroxide solution (0.012%-0.00012% final concentration) to yield the final hydrogel with desired rigidity.
- quantities of reagents provided in this paragraph could be scaled up appropriately by a person of ordinary skill in the art.
- Example 2 Example 2:
- the IX formulation contained a 1:1 stoichiometric ratio of EDC based on the quantity of CO 2 H groups present on the HA molecules in the reaction mixture.
- the 10X formulation contained a 10:1 stoichiometric ratio (or 10-fold excess) of EDC based on the quantity of CO 2 H groups present on the HA molecules in the reaction mixture.
- a stoichiometric excess of tyramine was provided relative to the quantity of CO 2 H groups on HA.
- the reactants and the appropriate amount of EDC for the formulation were combined in a vial and agitated to facilitate the tyra ine-substitution reaction.
- Tyramine exhibits a UV absorbance peak at 275 nm, making the degree of tyramine substitution easily detectible against a tyramine calibration curve. Based on UV- spectroscopic analysis of the above three T-HA formulations, it was discovered that the HA- tyramine substitution reaction carried out with no EDC present (formulation OX) resulted in substantially zero tyramine substitution onto the HA molecules. This confirmed the importance of using a carbodiimide reaction pathway in the tyramine substitution reaction.
- the tyramine absorption in the T-HA formulation prepared using a 1 : 1 EDC:CO 2 H stoichiometric ratio in the tyramine substitution reaction resulted in a tyramine substitution rate of about 1.7% relative to all available CO 2 H groups on the HA chains.
- the 10X formulation (10:1 EDC:CO 2 H ratio) resulted in about a 4.7% substitution rate.
- the dityramine structure exhibits a blue fluorescence on exposure to UV light.
- the products of each of the above formulations were exposed to UV light to detect the presence of dityramine cross-links.
- both the IX and 10X hydrogels exhibited blue fluorescence (the 10X hydrogel fluorescence being more intense than that of the IX hydrogel), while the OX formulation exhibited no blue fluorescence at all.
- HA is a highly ubiquitous material in the body, and is non-immunogenic in humans.
- the invented cross-linked macromolecular network comprised a majority of unaltered native HA, will have substantial application in a wide variety of tissue engineering applications where it is desirable or necessary to provide synthetic tissue in a human body.
- tissue engineering applications where it is desirable or necessary to provide synthetic tissue in a human body.
- a high degree of tyramine substitution e.g. greater than about 10-20%, may be undesirable; the above described experiments demonstrated that such high degrees of substitution are unnecessary to provide a suitable T- HA network.
- a dihydroxyphenyl e.g.
- dityramine) cross-linked polycarboxylate (e.g. HA) network has a hydroxyphenyl (tyramine) substitution rate of less than 50, preferably less than 40, preferably less than 30, preferably less than 20, preferably less than 15, preferably less than 10, preferably less than 9, preferably less than 8, preferably less than 7, preferably less than 6, preferably less than 5, percent based on the total quantity of CO 2 H groups present on the polycarboxylate (HA) molecules.
- Each of the above preparations was then reacted in the presence of hydrogen peroxide and horseradish peroxidase, also as in Example 1, to form dityramine cross-links between the T-HA molecules and provide respectively Hydrogels 1, 2, 3, 4 and 5.
- Each of these five hydrogels was found, surprisingly and unexpectedly, to be a stable and substantially coherent material with the physical properties of each hydrogel varying relative to the concentration of T-HA in the preparation from which it was made.
- qualitatively Concentration 1 resulted in Hydrogel 1 having rigidity and rheological properties comparable to that of Vaseline or jelly; the hydrogel was stable and coherent yet could be caused to flow or spread on application of an external force, e.g. from a spatula or other conventional tool.
- Hydrogel 1 exhibited excellent adhesive properties making it an ideal candidate for a nonallergenic coating material for surgical instruments during surgery, e.g. ophthalmologic surgery.
- Hydrogel 2 was more rigid than Hydrogel 1 due to the greater concentration of T-HA in the preparation from which it was made, and the consequent predicted decrease in intramolecular cross-linking and increase in intermolecular cross-linking associated with increased T-HA concentration.
- Hydrogel 2 exhibited rheological and rigidity properties characteristic of gelatins, with a degree of viscoelastic reboundability on external loading. On greater loading, Hydrogel 2 was found to break up into smaller pieces instead of flowing, also characteristic of a gelatinous material.
- Hydrogel 3 had the properties and consistency of a dough or malleable paste, also not flowing on application of an external loading force. This material also exhibited substantially greater viscoelastic properties compared to Hydrogels 1 and 2.
- Hydrogel 4 was a highly rigid and coherent gel that strongly resisted breaking up on application of an external loading force. Hydrogel 4 was a highly resilient rubber-like composition that actually generated substantial springing force upon sudden compression (e.g. dropping onto the floor). This ability of Hydrogel 4 to generate such a springing force in response to a sudden compression may make this material ideal for certain joint replacement/repair applications where the joint undergoes repeated and periodic compressional loading (e.g. the ankle joint).
- Hydrogel 5 had cartilage-like properties with both the appearance of articular cartilage and the feel of cartilage upon cutting with a surgical blade.
- Confined compression tests were performed to quantitatively dete ⁇ nine the compressive mechanical properties of the five different hydrogels described above.
- a custom built polycarbonate confining chamber, and porous polypropylene filter platen (20 ⁇ m pores, 20% porosity) were used to perform the confined compression testing.
- Five cylindrical plugs (7.1 mm in diameter, approximately 3 mm in thickness) at each hydrogel concentration were made using the confining chamber and the freeze-thaw technique described in Example 4 below. The following testing protocol was followed for a series of stress relaxation tests in confined compression. All testing was performed using an Instron 5543 machine under computer control, which recorded the time-displacement-load data at a frequency of 10 Hz.
- a +5 N or +50 N load cell (Sensotec) was used to monitor load throughout each test.
- the thickness of each sample tested in confined compression was determined mechanically, by measuring the displacement at which the compressive response initiated relative to the bottom of the chamber as measured with the Instron 5543 machine. The measured thickness was used to calculate the strain percentage for each step.
- the compressive mechanical properties of the five hydrogels were determined as described in the preceding paragraph. Load data was normalized by sample cross-sectional area (39.6 mm 2 ) to compute stress. The equilibrium stress was plotted against the applied strain for each material formulation. The aggregate modulus at each step was defined as the equilibrium stress divided by the applied strain. For each material, the aggregate modulus was defined as the slope of the equilibrium stress-strain data in the most linear range. The results for the confined compression tests are shown in Fig. 4. All five hydrogels were testable in confined compression, and demonstrated characteristic stress relaxation responses typical of biphasic materials (such as cartilage).
- the aggregate moduli for the 6.25 mg/ml and 12.5 mg/ml T-HA hydrogels were 1-2 orders of magnitude lower than articular cartilage.
- the 25 mg/ml T-HA hydrogel displayed an aggregate modulus of approximately half of the reported values for articular cartilage.
- the 50 and 100 mg/ml T-HA hydrogels displayed aggregate moduli, which over the linear range were less than reported values of articular cartilage, but which had moduli that were larger than articular cartilage at strains of 15-20%.
- a dityramine cross-linked hyaluronan network will produce a coherent hydrogel material whose rigidity and other physical (rheological) properties can be tuned by varying the T-HA concentration prior to cross-linking the tyramine groups to suit a particular application.
- the coherence and elastic properties of these hydrogels was observed even absent any (or substantially any) SO 2" groups in the network to supply the charge-to-charge repulsive forces to generate the material's compression resistance and elasticity. This was a highly surprising and unexpected result that may have substantial positive consequences in tissue engineering applications.
- Hyaluronan is a highly ubiquitous and non-immunogenic molecule found in humans.
- hydrogels consisting of dityramine cross-linked hyaluronan networks may provide very suitable tissue replacement materials that can be implanted within a human body, whose rigidity can be tuned based on the application.
- these materials will be composed of predominantly unaltered hyaluronan which is non-immunogenic, and it is believed may result in zero or substantially zero immune response. This is an important advantage over many conventional tissue engineered materials whose formation chemistries prevent their appUcation in vivo due to harsh reaction conditions or reagents, and whose final chemical structures are more likely to induce an immune response.
- a number of methods of preparing hydrogels such as those described in Example 3 have been developed to cast or form the hydrogel into a predetermined three-dimensional shape. This is important for myriad tissue engineering appUcations where it is necessary to provide artificial tissue material to fill a native tissue defect or void within a patient.
- a first method is to employ an in situ forming technique where the hydrogel is formed in place, i.e. in position and in the shape of its final appUcation and structure.
- the in situ formation method has been carried out experimentally as foUows.
- Tyramine-substituted hyaluronan (T-HA) was prepared via the carbo ⁇ mide-mediated pathway described herein. Following dialyzation to remove unreacted tyramine, EDC, NHS, etc., and dissolution at the desired concentration in PBS (see Example 1 above), a small quantity of horseradish peroxidase enzyme was added to the T-HA liquid preparation to form a first solution.
- This first solution was provided into a laboratory container (to simulate an in vivo situs) having a specific interior geometry. Subsequently, a second solution was prepared containing very dilute hydrogen peroxide (0.012%-0.00012% final concentration). A small volume of this second solution relative to the first solution was then injected into the container already containing the first solution to initiate the dityramine cross-Unking reaction to yield the hydrogel. Hydrogels prepared by this technique have been prepared having varying rigidity and rheological properties as described above in Example 3, and conformed weU to the interior surface contour of the container in which they were formed.
- a second method is a porous mold technique and is suitable for forming hydrogels into more complex three-dimensional structures.
- a porous hoUow mold is first cast conforming to the shape and contour of the intended final structure.
- a mold can be prepared having an interior surface in a cuboid shape if a cuboid shaped hydrogel were desired.
- the mold can be prepared or cast via conventional techniques from conventional porous materials, e.g. plaster of paris, porous or sintered plastics or metals, etc.
- the mold is prepared using a cellulosic dialysis membrane.
- the first and second solutions are prepared as above, and the first solution is provided into the hollow mold cavity of the porous mold.
- the now-filled mold is submersed in a bath of very dilute peroxide.
- the macromolecular T-HA and peroxidase molecules are unable to diffuse out of the porous mold due to their size, however the very small peroxide molecules (H 2 O 2 ) are able to diffuse in and react in the presence of the peroxidase enzyme to yield dityramine cross-links.
- H 2 O 2 very small peroxide molecules
- cross- linking occur from the outside inward to produce the finished hydrogel shape, and a certain degree of trial and error may be required to determine optimal or sufficient immersion times in the peroxide bath. Determination of these time periods is within the skill of a person having ordinary skill in the art.
- Successfully completed three-dimensional hydrogel shapes have been prepared in laboratory bench experiments via this porous mold technique.
- a third method is a freeze-thaw technique that is suitable for casting hydrogels according to the invention in highly intricate predetermined three-dimensional shapes, e.g. having internal folds such as a human ear.
- a mold is prepared from a soft or malleable material such as a polymeric material having a low glass transition temperature, e.g. below -80°C.
- the preferred mold materials are silicones having low glass transition temperatures, such as polydimethylsiloxane whose glass transition temperature is about - 127°C, however other suitably low glass transition (e.g. below -80°C) silicones, as well as other polymers, can be used.
- the silicone (preferred material) is first prepared such that it has an inner mold cavity conforming to the surface shape, contour and volume of a desired hydrogel part via any conventional or suitable technique (i.e. press-molding, carving, etc.).
- First and second solutions are prepared as above, and the first solution is provided into the inner mold cavity of the silicone mold.
- the now-filled siUcone mold is then cooled to about -80°C by contacting with soUd CO 2 (dry ice). Because the first solution is principally water, it freezes into a soUd ice form conforming to the shape and contour of the inner mold surface.
- the silicone mold having a glass transition temperature below -80°C, remains soft and malleable and the soUd ice form of the first solution is easily removed.
- suitable mechanical hardware should be used to ensure the silicone mold does not deform or expand as the solution freezes.
- port holes are provided in the mold to allow for expansion and discharge of the first solution as it expands during the freezing process.
- the threshold glass transition temperature of -80°C for the mold material is selected to correspond roughly with the surface temperature of soUd CO 2 (dry ice), to ensure the mold material does not become brittle when the first solution is frozen to produce the solid ice form.
- the threshold glass transition temperature for suitable mold materials may be adjusted accordingly.
- the first solution contained both the peroxidase and T-HA, while the second solution contained the peroxide. While it may be possible to switch the peroxidase and peroxide in the first and second solutions respectively, it is less preferred to provide the peroxide in the first solution with the T-HA. This is because once the peroxide, peroxidase and T-HA are combined, the T-HA rapidly begins to form a cross-linked macromolecular network.
- the peroxidase (which is a macromolecular molecule) is not already uniformly distributed with the T-HA it may be unable or substantially hindered from diffusing through the pore structure of the forming hydrogel to facilitate uniform cross-linking throughout the entire T-HA/peroxide solution. The result could be non-uniform and/or incomplete cross-Unking of the T-HA and a non- uniform hydrogel.
- the relatively small peroxide molecule hydrogen peroxide is only one oxygen atom larger than water
- the macromolecular size of the peroxidase allows it to be similarly retained as the T-HA within porous molds that are only porous to small molecular weight peroxides which easily and uniformly diffuse through both the molds and newly forming macromolecular networks (i.e. hydrogels). For these reasons it is preferred to start with the peroxidase uniformly distributed with the T-HA in the first solution, and to provide the peroxide separately in the second solution.
- a fourth method is an alternating sprayed or brushed layering technique.
- the first solution is prepared as described above and contains both the peroxidase and T-HA.
- the second solution not only contains the peroxide as described above, but also T- HA at the same concentration as in the first solution.
- a thin layer of the first solution is apphed at the desired location (in situ) followed by an overlying thin layer of the second solution. This procedure is repeated such that alternating layers of the first and second solutions are successively applied until the defect or appUcation situs has been completed.
- the very thin alternating layers of the first and second solutions promote virtually complete dityramine cross-Unking ensuring a highly coherent final hydrogel having the desired rheological properties based on the initial T-HA concentration of the two solutions.
- the thin nature of the layers is desirable to ensure that free radicals produced by the peroxidase in the first solution layers are able to penetrate completely adjacent second solution layers and complete cross-linking independent of peroxidase diffusion into the second solution layer (see above).
- T-HA is included in both solutions to ensure uniform T-HA concentration throughout the final hydrogel. This technique has been performed in laboratory bench experiments and has provided contour-conforming and volume-filling coherent hydrogels.
- This technique is highly applicable where it is desired to provide a thin, but variable layer of tyramine cross-linked HA, such as on the surface of a denuded osteoarthritic joint in which Uttle if any native healthy cartilage remains in the patient at the implant site.
- Rat chondrocytes were embedded in (cross-linked) T-HA hydrogels to measure their ability to survive the cross-linking reaction. Isolated chondrocytes were suspended in the 1.7% and 4.7% T-HA hydrogels described in Example 2 by providing these Uve cells to the first solution to be co-dispersed with the T-HA and peroxidase, followed by introduction of the peroxide-containing second solution to initiate dityramine cross-linking. The chondrocyte-embedded 1.7% and 4.7% T-HA hydrogels exhibited uniformly distributed chondrocytes with the optical clarity of the gels allowing visualization throughout the gel.
- Glucose utilization was used as an indicator of cell viability after cross-linking to form the hydrogels as chondrocytes are voracious with respect to glucose consumption, depleting the medium of glucose in less than 24 hours.
- the results showed that chondrocytes embedded in T-HA hydrogels showed essentially the same glucose consumption profile over 24 hours as the same chondrocytes cultured in monolayer (Fig. 5). This continued for up to 7 days indicating that the cells were alive and metaboUcally active.
- Medium glucose was measured by standard hexokinase assay.
- HA samples from both the hydrogel scaffold and cartilage matrix were visualized by fluorescent staining with biotinylated HA binding protein (b-HABP) reagent while cell nuclei were visualized with standard DAPI stain.
- b-HABP reagent is prepared from purified cartilage aggrecan (the Gl domain only) and link protein, and recognizes and irreversibly binds to stretches of native HA equivalent to those normally bound by aggrecan and link protein in cartilage.
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- 2004-01-09 CN CNA2008100880491A patent/CN101366974A/en active Pending
- 2004-01-09 CN CNB2004800055719A patent/CN100467538C/en not_active Expired - Fee Related
- 2004-01-09 WO PCT/US2004/000478 patent/WO2004063388A2/en active Application Filing
- 2004-01-09 CA CA2512730A patent/CA2512730C/en not_active Expired - Fee Related
- 2004-01-09 AU AU2004204465A patent/AU2004204465B2/en not_active Ceased
- 2004-01-09 JP JP2006500869A patent/JP5325385B2/en not_active Expired - Fee Related
- 2004-01-09 EP EP04701177.0A patent/EP1587945B1/en not_active Expired - Lifetime
-
2005
- 2005-08-05 US US11/198,803 patent/US7368502B2/en active Active
Non-Patent Citations (2)
Title |
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See also references of WO2004063388A2 |
SOFIA S.J. ET AL.: "PEROXIDASE-CATALYZED CROSSLINKING OF FUNCTIONALIZED POLYASPARTIC ACID POLYMERS", JOURNAL OF MACROMOLECULAR SCIENCE, vol. A39, no. 10, 2002, pages 1151 - 1181, XP009029356 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021043349A1 (en) * | 2019-09-06 | 2021-03-11 | Contipro A.S. | Hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid |
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WO2004063388A3 (en) | 2005-09-01 |
CA2512730C (en) | 2013-04-02 |
CA2512730A1 (en) | 2004-07-29 |
AU2004204465A2 (en) | 2004-07-29 |
US7368502B2 (en) | 2008-05-06 |
CN100467538C (en) | 2009-03-11 |
US20040147673A1 (en) | 2004-07-29 |
JP2006517598A (en) | 2006-07-27 |
US6982298B2 (en) | 2006-01-03 |
US20050265959A1 (en) | 2005-12-01 |
AU2004204465B2 (en) | 2009-11-19 |
CN1826381A (en) | 2006-08-30 |
EP1587945A4 (en) | 2006-03-22 |
EP1587945B1 (en) | 2017-11-01 |
CN101366974A (en) | 2009-02-18 |
WO2004063388A2 (en) | 2004-07-29 |
AU2004204465A1 (en) | 2004-07-29 |
JP5325385B2 (en) | 2013-10-23 |
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